In laser scanning imaging systems, such as microscopes or macroscopes, an incident laser beam is focused on a specimen and the focal spot is scanned through the specimen. In conventional laser scanning imaging systems, the incident laser beam is focused by an objective lens to a diffraction-limited spot on or within the specimen, which may or may not be fluorescent. Scattered and reflected laser light as well as any re-emitted (i.e. fluorescent) light emanating from the illuminated spot is collected by the objective lens, and separated from the incident beam by one or more beam splitters. A photo-detector transforms this light signal emanating from the sample into an electrical signal which is recorded by a computer. The detected light originating from one illuminated diffraction-limited spot of the specimen represents one pixel in the resulting image. As the laser scans over the specimen, a whole image is obtained pixel by pixel, in which the brightness of each pixel corresponds to the relative intensity of detected light.
By way of example, FIG. 1 (PRIOR ART) shows an example of a laser scanning imaging system 20 used for two-photon excited fluorescence. The laser scanning imaging system 20 includes a laser module 22 for generating a laser beam and an imaging device 24 (i.e. laser scanning microscope) adapted to focus the laser beam into a diffraction-limited spot size within or on the surface of a sample 30, and to collect any light emanating from the sample 30 as a result of the probing by the laser beam. As known in the art, the laser scanning microscope generally includes imaging optics 32 such as an objective lens 34 and a beam splitter 36, an image sensor 38 sensing light emanating from the sample 30 upon being probed by the laser beam, as well as a scanning module 48 (e.g. a scan head) adapted to scan the laser beam over the sample 30.
Laser scanning microscopy methods (e.g. confocal, two-photon or multi-photon microscopy) are usually preferred to wide-field microscopy methods for their z-sectioning ability. For example, confocal and two-photon laser scanning fluorescence microscopes having better spatial resolution than conventional wide-field microscopes are now commonly employed for imaging narrow sections of biological structures, in which molecules of interest are tagged with fluorescent markers. Indeed, both confocal and two-photon laser microscopes can provide depths of field of the order of only a few microns, which leads to excellent optical sectioning capabilities. This key feature of laser scanning microscopy allows acquiring multiple in-focus images of thin sections at selected depths within a sample and therefore enables three-dimensional imaging of thick samples. However, the transverse resolution of laser scanning microscopy remains similar to that of wide-field microscopy.
Microscopy is generally limited in resolution by the diffraction barrier also known as the Abbe or Rayleigh limit. In theory, this limit is λ/2, where λ is the optical wavelength of the light used to probe the material being investigated. In practice, however, this limit can only be reached with optimized high-numerical aperture instruments. For biomedical or material applications, high resolution is often needed and a large variety of methods have been developed to overcome this limit. Methods developed for enhancing the resolution of microscopes are often referred to as “super-resolution” or “hyper-resolution” methods.
Super-resolution methods can be classified into three categories. The first category relies on optical shaping of the excitation volume and includes the stimulated emission depletion (STED) microscopy developed by Stefan Hell. The STED approach is based on the depletion of fluorescence emission in a ring around the focal point using stimulated emission, triplet-state shelving, or reversible saturable optical fluorescence transitions (RESOLFT) [S. W. Hell et al., “Breaking the diffraction resolution limit by stimulated emission: stimulated emission depletion microscopy”, Opt. Lett. vol. 19, pp. 780-782 (1994); S. W. Hell, “Process and device for optically measuring a point on a sample with high local resolution”, U.S. Pat. No. 5,731,588 (1998); S. Bretschneider et al., “Breaking the diffraction barrier in fluorescence microscopy by optical shelving”, Phys. Rev. Lett. vol. 98, pp. 218103 (2007); M. Hofmann et al., “Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins”, Proc. Natl. Acad. Sci. USA vol. 102, pp. 17565-17569 (2005)]. A strong increase in resolution is obtained using this technique, but high peak power lasers, which can cause photobleaching and possibly photodamage, or specific probes (e.g. molecular absorbers/emitters) are needed. A STED macroscope would require the use of a high power laser to get enough intensity at the focus for the depletion, because the spot size is significantly larger in macroscopy compared to microscopy. Furthermore, STED cannot be retrofitted into an existing laser scanning microscope and is limited to fluorescence imaging. Complex multi-color confocal and single-color two-photon versions of STED exist, but they are more restrictive on the probe selection compared to conventional multi-color confocal and two-photon microscopes [J. Bückers et al., “Simultaneous multi-lifetime multi-color STED imaging for colocalization analyses”, Opt. Express vol. 19, pp. 3130-3143 (2011); J. B. Ding et al., “Supraresolution imaging in brain slices using stimulated-emission depletion two-photon laser scanning microscopy”, Neuron vol. 63, pp. 429-437 (2009)].
The second category relies on single molecule imaging and localization. It includes photo-activation localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), and many other methods based on active control of the emitting molecules using photo-activation or photo-switching [H. Shroff et al., “Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes”, Proc. Natl. Acad. Sci. USA vol. 104, pp. 20308-20313 (2007); M. Bates et al., “Multicolor super-resolution imaging with photo-switchable fluorescent probes”, Science vol. 317, pp. 1749-1753 (2007); B. Huang et al., “Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy”, Science vol. 319, pp. 810-813 (2008); X. Zhuang et al., “Sub-diffraction image resolution and other imaging techniques” U.S. Pat. No. 7,776,613]. A high accuracy of molecule position is obtained using these methods, but specific probes and much longer acquisition times are needed. These methods are also subject to mathematical artifacts since they rely on calculations of the centroid of the diffraction spot. These methods are often applied to total internal reflectance fluorescence (TIRF) microscopy and oblique illumination microscopy, and their working distance is very limited.
The third category of super-resolution methods is referred to as “structured illumination” and allows improving the resolution of wide-field microscopes [P. Kner et al., “Super-resolution video microscopy of live cells by structured illumination”, Nature Methods vol. 6, pp. 339-342 (2009)]. In brief, structured illumination consists in exciting fluorescent species in a sample using a beam made of periodic parallel lines produced by the interference between two laser beams. Multiple images of the sample are taken at different orientations and phases of the periodic pattern. Data acquisition is followed by sophisticated image processing in order to generate super-resolved images. A gain in resolution by a factor of two is obtained compared to conventional imaging systems, and resolution can be further enhanced if nonlinearity can be exploited.
Other approaches for enhancing resolution in laser scanning microscopy that have been proposed include works by B. R. Boruah [B. R. Boruah, “Lateral resolution enhancement in confocal microscopy by vectorial aperture engineering”, Applied Optics vol. 49, pp. 701-707 (2010)] and O. Haeberlé and B. Simon [O. Haeberlé and B. Simon, “Saturated structured confocal microscopy with theoretically unlimited resolution”, Optics Communications vol. 282, pp. 3657-3664 (2009)].
Commercial products based on STORM, on STED and on structured illumination have been put on the market. These instruments are specified for a resolution down to 20 nm (STORM), 50 nm (STED) and 100 nm (structured illumination). However, the cost of these systems is significantly higher than that of most current wide-field and laser scanning microscopes. Moreover, all of the above-mentioned super-resolution methods inherently rely on fluorescence and on specific photophysical properties of the fluorescent molecular probes.
There therefore remains a need for improving the resolution of laser scanning microscopic systems in a practical and cost effective manner.